Anti-SSX-2 T cell receptors and related materials and methods of use

ABSTRACT

The invention provides an isolated or purified T cell receptor (TCR) having antigenic specificity for synovial sarcoma X Breakpoint (SSX)-2. The invention further provides related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, and populations of cells. Further provided by the invention are antibodies, or an antigen binding portion thereof, and pharmaceutical compositions relating to the TCRs of the invention. Methods of detecting the presence of cancer in a host and methods of treating or preventing cancer in a host are further provided by the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is the U.S. national phase of InternationalPatent Application No. PCT/US2011/051537, filed Sep. 14, 2011, whichclaims the benefit of U.S. Provisional Patent Application No.61/384,931, filed Sep. 21, 2010, each of which is incorporated byreference in its entirety herein.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 41,491 Byte ASCII (Text) file named“712297ST25.TXT,” dated Feb. 14, 2013.

BACKGROUND OF THE INVENTION

Adoptive cell therapy (ACT) involves the transfer of reactive T cellsinto patients, including the transfer of tumor-reactive T cells intocancer patients. Adoptive cell therapy has been successful in causingthe regression of tumors in some cancers, e.g., melanoma. One obstacleto the widespread application of adoptive cell therapy is the difficultyin generating human T cells with anti-tumor potential. Another obstacleto the successful application of adoptive cell therapy is that thetransferred T cells can also be toxic to normal, i.e., non-canceroustissues. Accordingly, there exists a need for improved immunologicalcompositions and methods for treating cancer.

BRIEF SUMMARY OF THE INVENTION

The invention provides an isolated or purified T cell receptor (TCR)having antigenic specificity for synovial sarcoma X Breakpoint (SSX)-2(SEQ ID NO: 1). The TCR can comprise specified amino acid sequences asdescribed herein. For instance, the inventive TCR can comprise the aminoacid sequence of any one or more of SEQ ID NOs: 13-18, SEQ ID NOs: 19and 20, SEQ ID NOs: 23 and 24, or SEQ ID NOs: 25 and 26.

The invention further provides related polypeptides and proteins, aswell as related nucleic acids, recombinant expression vectors, hostcells, and populations of cells. Further provided by the invention areantibodies, or an antigen binding portion thereof, and pharmaceuticalcompositions relating to the TCRs of the invention.

Methods of detecting the presence of cancer in a host and methods oftreating or preventing cancer in a host are further provided by theinvention. The inventive method of detecting the presence of cancer in ahost comprises (i) contacting a sample comprising cells of the cancerwith any of the inventive TCRs, polypeptides, proteins, nucleic acids,recombinant expression vectors, host cells, populations of host cells,or antibodies, or antigen binding portions thereof, described herein,thereby forming a complex, and (ii) detecting the complex, whereindetection of the complex is indicative of the presence of cancer in thehost.

The inventive method of treating or preventing cancer in a hostcomprises administering to the host any of the TCRs, polypeptides, orproteins described herein, any nucleic acid or recombinant expressionvector comprising a nucleotide sequence encoding any of the TCRs,polypeptides, proteins described herein, or any host cell or populationof host cells comprising a recombinant vector which encodes any of theTCRs, polypeptides, or proteins described herein, in an amount effectiveto treat or prevent cancer in the host.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a bar graph showing interferon-γ (IFN-γ) levels measuredafter SSX-2 TCR-transduced peripheral blood leukocytes (PBLs) wereco-cultured with T2 cells from a human donor, wherein the T2 cells werepulsed with varying concentrations of the SSX-2: 41-49 (KASEKIFYV) (SEQID NO: 2) peptide.

FIG. 1B is a bar graph showing IFN-γ levels measured, as in FIG. 1A,except the T2 cells were from a different human donor.

FIG. 2 is a bar graph showing IFN-γ levels measured when SSX-2TCR-transduced PBLs were co-cultured with 293-A2 and COST-A2 cellsexpressing the SSX-2 gene and T2 cells pulsed with the SSX-2: 41-49peptide.

FIG. 3 is a bar graph that shows the resulting IFN-γ levels measuredwhen SSX-2 TCR-transduced PBLs (shaded bars) or untransduced (UT) PBLs(unshaded bars) were co-cultured with various tumor cell lines.

FIG. 4A is a bar graph that shows IFN-γ levels after PBLs from a humandonor, that were transduced with a SSX-2 TCR (shaded bars) or nottransduced (UT) (unshaded bars), were co-cultured with various tumorcells.

FIG. 4B is a bar graph that shows IFN-γ levels, as in FIG. 4A, exceptthe PBLs were from a different human donor.

FIG. 5 is a line graph that shows the results of co-culture assaysperformed with SSX-2 TCR transduced-PBLs and peptide-pulsed T2 cells.The peptides used for pulsing were: SSX-1 (KYSEKISYV, SEQ ID NO: 32)(-♦-); SSX-2 (KASEKIFYV, SEQ ID NO: 2) (▪); SSX-3 (KVSEKIVYV, SEQ ID NO:8) (▴); SSX-4 (KSSEKIVYV, SEQ ID NO: 9) (X); SSX-5 (KASEKIIYV, SEQ IDNO: 10) (*); SSX-6 (KFSEKISCV, SEQ ID NO: 33) (●); SSX-7 (KSLEKISYV, SEQID NO: 34) (|); SSX-8 KYSEKISYV, SEQ ID NO: 32) (-); SSX-9 (KSSEKIIYV,SEQ ID NO: 11) (—); and SSX-10 (KASEKILYV, SEQ ID NO: 12) (--♦--).

FIGS. 6A and 6B are bar graphs showing proliferation (in terms of[³H]-thymidine incorporation counts per minute (CPM)) of PBLs from Donor1 (A) or Donor 2 (B) that were untransduced (UT) or transduced withSSX-2 TCR (“SSX-WT”), codon-optimized SSX-2 TCR (“SSX-CO op”), or acodon-optimized human-mouse chimera SSX-2 TCR (“SSX-MCR”).

FIGS. 7A-D are graphs showing percent lysis of 938 mel (HLA-A2−/SSX-2+)(A), COS-A2 (B), 938-A2 mel (C), COS-A2-SSX-2 (D) when co-cultured withPBL that were untransduced (♦) or transduced with SSX-2 TCR (▪),codon-optimized SSX-2 TCR (▴), or a codon-optimized human-mouse chimeraSSX-2 TCR (X) at the indicated effector to target (E:T) ratios.

FIGS. 8A-D are line graphs showing percent lysis of 624 mel (A), 1300mel (B), SK mel 37 (C), or 888 mel (D) when co-cultured with PBL thatwere untransduced (♦) or transduced with SSX-2 TCR (▪), codon-optimizedSSX-2 TCR (▴), or a codon-optimized human-mouse chimera SSX-2 TCR (X) atthe indicated effector to target (E:T) ratios.

FIGS. 9A-9E are bar graphs showing proliferation (in terms of[³H]-thymidine incorporation counts per minute (CPM)) of PBLs that wereuntransduced or transduced with SSX-2 TCR (“SSX-TCR-WT”),codon-optimized SSX-2 TCR (“SSX-TCR-codon optimized-PBL”), or acodon-optimized human-mouse chimera SSX-2 TCR (“SSX-2-TCR mouse constantregion-PBL”).

FIG. 10 is a bar graph showing IFN-γ levels measured after PBLstransduced with SSX-2 TCR (“SSX2-WT”) (grey bars), codon-optimized SSX-2TCR (“SSX2-Co Op”) (unshaded bars), or a codon-optimized human-mousechimera SSX-2 TCR (“SSX2-MCR”) (black bars) were co-cultured with T2cells from a human donor, wherein the T2 cells were pulsed with varyingconcentrations of the SSX-2: 41-49 peptide.

FIG. 11 is a bar graph that shows IFN-γ levels after PBLs from a humandonor, that were transduced with a SSX-2 TCR (unshaded bars) or nottransduced (UT) (shaded bars), were co-cultured with various primarymelanoma cells.

FIG. 12 is a bar graph that shows IFN-γ levels after PBLs from a humandonor that were transduced with a SSX-2 TCR were co-cultured withmel1300 cells in the absence (grey bars) or in the presence (0.1 μM(unshaded bars) or 1.0 μM (black bars)) of the demethylating agent,5-aza-2′-deoxycytidine (DAC). NM is normal media with no drug control.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides an isolated or purified T cell receptor (TCR)having antigenic specificity for synovial sarcoma X breakpoint (SSX)-2(also known as HOM-MEL-40). SSX-2 is a member of the SSX family of tenhighly homologous nuclear proteins also including SSX-1, SSX-3, SSX-4,SSX-5, SSX-6, SSX-7, SSX-8, SSX-9, and SSX-10. The SSX proteins arecancer testis antigens (CTA), which are expressed only in tumor cellsand non-MHC expressing germ cells of the testis. SSX-2 is expressed in avariety of human cancers including, but not limited to, melanomas, headcancers, neck cancers, lymphomas, multiple myeloma, pancreatic cancer,prostate cancer, sarcomas, hepatocellular and colon carcinomas. TheSSX-2 protein may comprise, consist, or consist essentially of, SEQ IDNO: 1.

The phrase “antigenic specificity” as used herein means that the TCR canspecifically bind to and immunologically recognize SSX-2 with highavidity. For example, a TCR may be considered to have “antigenicspecificity” for SSX-2 if T cells expressing the TCR secrete at leastabout 200 pg/ml or more (e.g., 200 pg/ml or more, 300 pg/ml or more, 400pg/ml or more, 500 pg/ml or more, 600 pg/ml or more, 700 pg/ml or more)of IFN-γ upon co-culture with a low concentration of HLA-A2 restrictedSSX-2 (e.g., about 0.01 ng/ml to about 1 ng/ml, 0.01 ng/ml, 0.1 ng/ml,or 1 ng/ml). The inventive TCRs may also secrete IFN-γ upon co-culturewith higher concentrations of SSX-2.

An embodiment of the present invention includes an isolated or purifiedT cell receptor (TCR) having antigenic reactivity toward synovialsarcoma X Breakpoint (SSX)-2 or SSX-2 and any one or more of SSX-3,SSX-4, SSX-5, SSX-9, and SSX-10.

The TCR may have antigenic specificity for any SSX-2 protein,polypeptide or peptide. In an embodiment of the invention, the TCR hasantigenic specificity for an SSX-2 protein comprising, consisting of, orconsisting essentially of, SEQ ID NO: 1. In a preferred embodiment ofthe invention, the TCR has antigenic specificity for an SSX-2 peptidecomprising, consisting of, or consisting essentially of, KASEKIFYV (SEQID NO: 2).

While the TCRs of the invention have antigenic specificity for SSX-2,the TCRs of the invention can also recognize any one or more of SSX-3,SSX-4, SSX-5, SSX-9, and SSX-10. That is, the TCRs of the invention canbind to and immunologically recognize any one or more of SSX-3, SSX-4,SSX-5, SSX-9, and SSX-10, but with a lower avidity than that which isobserved for binding to SSX-2, such that the binding of the TCR to oneof these proteins elicits an immune response at a higher concentrationof any one of these proteins than that which is necessary to elicit animmune response with SSX-2. For example, the TCR of the invention may beconsidered to recognize any one or more of SSX-3, SSX-4, SSX-5, SSX-9,and SSX-10 with low avidity if T cells expressing the TCR do not secreteat least about 200 pg/ml (e.g., secretes less than 200 pg/ml, less than100 pg/ml) of IFN-γ upon co-culture with a low concentration of any oneor more of SSX-3, SSX-4, SSX-5, SSX-9, and SSX-10 (e.g., about 0.01ng/ml to about 1 ng/ml, 0.01 ng/ml, 0.1 ng/ml, or 1 ng/ml) but dosecrete at least about 200 pg/ml or more (e.g., 200 pg/ml or more, 300pg/ml or more, 400 pg/ml or more, 500 pg/ml or more, 600 pg/ml or more,700 pg/ml or more) upon co-culture with a higher concentration of anyone or more of SSX-3, SSX-4, SSX-5, SSX-9, and SSX-10 (e.g., about 10ng/ml to about 100 ng/ml, 10 ng/ml, 50 ng/ml, or 100 ng/ml).

The TCR may recognize an SSX-3, SSX-4, SSX-5, SSX-9, and/or SSX-10protein, polypeptide or peptide. In an embodiment of the invention, theTCR recognizes a protein comprising, consisting of, or consistingessentially of, SEQ ID NO: 3 (SSX-3), SEQ ID NO: 4 (SSX-4), SEQ ID NO: 5(SSX-5), SEQ ID NO: 6 (SSX-9), and/or SEQ ID NO: 7 (SSX-10). In apreferred embodiment of the invention, the TCR recognizes a peptidecomprising, consisting of, or consisting essentially of, SSX-3 peptideKVSEKIVYV (SEQ ID NO: 8), SSX-4 peptide KSSEKIVYV (SEQ ID NO: 9), SSX-5peptide KASEKIIYV (SEQ ID NO: 10), SSX-9 peptide KSSEKIIYV (SEQ ID NO:11), and/or SSX-10 peptide KASEKILYV (SEQ ID NO: 12).

The inventive TCRs are able to recognize SSX-2, SSX-3, SSX-4, SSX-5,SSX-9, and/or SSX-10 (hereinafter, “SSX cancer antigens”) in anHLA-A2-dependent manner. By “HLA-A2-dependent manner” as used hereinmeans that the TCR elicits an immune response upon binding to an SSXcancer antigen within the context of an HLA-A2 molecule.

Furthermore, without being bound to any particular theory, the inventiveTCRs are able to recognize an SSX cancer antigen in a CD8- and/orCD4-independent manner. By “CD8- and/or CD4-independent manner,” ismeant that the inventive TCRs, upon binding to an SSX cancer antigen,can elicit an immune response in the absence of a CD8 or CD4 molecule,or both a CD8 and CD4 molecule, expressed on the cell expressing theinventive TCR or in the absence of a functional CD8 or CD4 molecule, orboth. Unlike traditional TCRs, the inventive TCRs do not have apreference for CD8 or CD4 and can function in the context of either aCD8 or CD4 molecule.

The TCRs of the invention provide many advantages, including when usedfor adoptive cell transfer. For example, without being bound by aparticular theory, it is believed that because SSX-2, SSX-3, SSX-4,SSX-5, SSX-9, and/or SSX-10 are expressed by cells of multiple cancertypes, the inventive TCRs advantageously provide the ability to destroycells of multiple types of cancer and, accordingly, treat or preventmultiple types of cancer. Additionally, without being bound to aparticular theory, it is believed that because the SSX proteins arecancer testis antigens that are expressed only in tumor cells andnon-MHC expressing germ cells of the testis, the inventive TCRsadvantageously target the destruction of cancer cells while minimizingor eliminating the destruction of normal, non-cancerous cells, therebyreducing, for example, minimizing or eliminating, toxicity. In addition,while the inventive TCRs have antigenic specificity for SSX-2, theinventive TCRs advantageously also recognize any one or more of SSX-3,SSX-4, SSX-5, SSX-9, and SSX-10. Without being bound to a particulartheory, it is believed that the ability to recognize multiple cancerantigens advantageously increases the number of cancer cells that can bedestroyed by the inventive TCRs. Additionally, should an SSX antigenbecome mutated, the inventive TCRs can still be viable in that theyrecognize more than just one antigen.

The invention provides a TCR comprising two polypeptides (i.e.,polypeptide chains), such as an α chain of a TCR, a β chain of a TCR, aγ chain of a TCR, a δ chain of a TCR, or a combination thereof. Suchpolypeptides chains of TCRs are known in the art. The polypeptides ofthe inventive TCR can comprise any amino acid sequence, provided thatthe TCR has antigenic specificity for SSX-2 and/or recognizes any one ormore of SSX-3, SSX-4, SSX-5, SSX-9, and SSX-10.

In an embodiment of the invention, the TCR comprises two polypeptidechains, each of which comprises a variable region comprising acomplementarity determining region (CDR) 1, a CDR2, and a CDR3 of a TCR.Preferably, the first polypeptide chain comprises a CDR1 comprising theamino acid sequence of SEQ ID NO: 13 (CDR1 of a chain), a CDR2comprising the amino acid sequence of SEQ ID NO: 14 (CDR2 of a chain),and a CDR3 comprising the amino acid sequence of SEQ ID NO: 15 (CDR3 ofa chain), and the second polypeptide chain comprises a CDR1 comprisingthe amino acid sequence of SEQ ID NO: 16 (CDR1 of β chain), a CDR2comprising the amino acid sequence of SEQ ID NO: 17 (CDR2 of β chain),and a CDR3 comprising the amino acid sequence of SEQ ID NO: 18 (CDR3 ofβ chain). In this regard, the inventive TCR can comprise the amino acidsequences selected from the group consisting of any one or more of SEQID NOs: 13-15, 16-18, and 13-18. Preferably the TCR comprises the aminoacid sequences of SEQ ID NOs: 13-18.

Alternatively or additionally, the TCR can comprise an amino acidsequence of a variable region of a TCR comprising the CDRs set forthabove. In this regard, the TCR can comprise the amino acid sequence ofSEQ ID NO: 19 (the variable region of an α chain) or 20 (the variableregion of a β chain), both SEQ ID NOs: 19 and 20, SEQ ID NO: 35 (aportion of the variable region of an α chain) or 36 (a portion of thevariable region of a β chain), or both SEQ ID NOs: 35 and 36.Preferably, the inventive TCR comprises the amino acid sequences of SEQID NOs: 19 and 20.

Alternatively or additionally, the TCR can comprise an α chain of a TCRand a β chain of a TCR. Each of the α chain and β chain of the inventiveTCR can independently comprise any amino acid sequence. Preferably, theα chain comprises the variable region of an α chain as set forth above.In this regard, the inventive TCR can comprise the amino acid sequenceof SEQ ID NO: 23. An inventive TCR of this type can be paired with any βchain of a TCR. Preferably, the β chain of the inventive TCR comprisesthe variable region of a β chain as set forth above. In this regard, theinventive TCR can comprise the amino acid sequence of SEQ ID NO: 24. Theinventive TCR, therefore, can comprise the amino acid sequence of SEQ IDNO: 23 or 24, or both SEQ ID NOs: 23 and 24. Preferably, the inventiveTCR comprises the amino acid sequences of SEQ ID NOs: 23 and 24.

In an embodiment of the invention, the TCR can comprise a human/mousechimeric TCR. In this regard, the TCR can comprise a mouse constantregion comprising SEQ ID NO: 21 (mouse constant region of an α chain),SEQ ID NO: 22 (mouse constant region of β chain), or both SEQ ID NOs: 21and 22. Preferably, the TCR comprises both SEQ ID NOs: 21 and 22.

Alternatively or additionally, the inventive human/mouse chimeric TCRcan comprise any of the CDRs set forth above. In this regard, theinventive human/mouse chimeric TCR can comprise the amino acid sequencesselected from the group consisting of SEQ ID NOs: 13-15, 16-18, and13-18. Preferably the human/mouse chimeric TCR comprises the amino acidsequences of SEQ ID NOs: 13-18.

Alternatively or additionally, the human/mouse chimeric TCR can compriseany of the variable regions set forth above. In this regard, theinventive human/mouse chimeric TCR can comprise the amino acid sequenceof SEQ ID NO: 19 (the variable region of an α chain) or 20 (the variableregion of a β chain), both SEQ ID NOs: 19 and 20, SEQ ID NO: 35 (aportion of the variable region of an α chain) or 36 (a portion of thevariable region of a β chain), or both SEQ ID NOs: 35 and 36.Preferably, the inventive TCR comprises the amino acid sequences of SEQID NOs: 19 and 20.

Alternatively or additionally, the human/mouse chimeric TCR can comprisean α chain of a TCR and a β chain of a TCR. Each of the α chain and βchain of the inventive human/mouse chimeric TCR can independentlycomprise any amino acid sequence. Preferably, the α chain comprises thevariable region of an α chain as set forth above. In this regard, theinventive human/mouse chimeric TCR can comprise the amino acid sequenceof SEQ ID NO: 25. An inventive human/mouse chimeric TCR of this type canbe paired with any β chain of a TCR. Preferably, the β chain of theinventive human/mouse chimeric TCR comprises the variable region of a βchain as set forth above. In this regard, the inventive human/mousechimeric TCR can comprise the amino acid sequence of SEQ ID NO: 26. Theinventive human/mouse chimeric TCR, therefore, can comprise the aminoacid sequence of SEQ ID NO: 25 or 26, or both SEQ ID NOs: 25 and 26.Preferably, the inventive TCR comprises the amino acid sequences of SEQID NOs: 25 and 26.

Also provided by the invention is an isolated or purified polypeptidecomprising a functional portion of any of the TCRs described herein. Theterm “polypeptide” as used herein includes oligopeptides and refers to asingle chain of amino acids connected by one or more peptide bonds.

With respect to the inventive polypeptides, the functional portion canbe any portion comprising contiguous amino acids of the TCR of which itis a part, provided that the functional portion specifically binds toSSX-2 and/or recognizes any one or more of SSX-3, SSX-4, SSX-5, SSX-9,and SSX-10. The term “functional portion” when used in reference to aTCR refers to any part or fragment of the TCR of the invention, whichpart or fragment retains the biological activity of the TCR of which itis a part (the parent TCR). Functional portions encompass, for example,those parts of a TCR that retain the ability to specifically bind toSSX-2 and/or recognize any one or more of SSX-3, SSX-4, SSX-5, SSX-9,and SSX-10 (e.g., in an HLA-A2-dependent manner), or detect, treat, orprevent cancer, to a similar extent, the same extent, or to a higherextent, as the parent TCR. In reference to the parent TCR, thefunctional portion can comprise, for instance, about 10%, 25%, 30%, 50%,68%, 80%, 90%, 95%, or more, of the parent TCR.

The functional portion can comprise additional amino acids at the aminoor carboxy terminus of the portion, or at both termini, which additionalamino acids are not found in the amino acid sequence of the parent TCR.Desirably, the additional amino acids do not interfere with thebiological function of the functional portion, e.g., specificallybinding to SSX-2; recognizing any one or more of SSX-3, SSX-4, SSX-5,SSX-9, and SSX-10; having the ability to detect cancer, treat or preventcancer, etc. More desirably, the additional amino acids enhance thebiological activity, as compared to the biological activity of theparent TCR.

The polypeptide can comprise a functional portion of either or both ofthe α and β chains of the TCRs of the invention, such as a functionalportion comprising one of more of CDR1, CDR2, and CDR3 of the variableregion(s) of the α chain and/or β chain of a TCR of the invention. Inthis regard, the polypeptide can comprise a functional portioncomprising the amino acid sequence of SEQ ID NO: 13 (CDR1 of a chain),14 (CDR2 of α chain), 15 (CDR3 of α chain), 16 (CDR1 of β chain), 17(CDR2 of β chain), 18 (CDR3 of β chain), or a combination thereof.Preferably, the inventive polypeptide comprises a functional portioncomprising SEQ ID NOs: 13-15, 16-18, or all of SEQ ID NOs: 13-18. Morepreferably, the polypeptide comprises a functional portion comprisingthe amino acid sequences of SEQ ID NOs: 13-18.

Alternatively or additionally, the inventive polypeptide can comprise,for instance, the variable region of the inventive TCR comprising acombination of the CDR regions set forth above. In this regard, thepolypeptide can comprise the amino acid sequence of SEQ ID NO: 19 (thevariable region of an α chain), 20 (the variable region of a β chain),both SEQ ID NOs: 19 and 20, SEQ ID NO: 35 (a portion of the variableregion of an α chain) or 36 (a portion of the variable region of a βchain), or both SEQ ID NOs: 35 and 36. Preferably, the polypeptidecomprises the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 20, orthe amino acid sequences of both SEQ ID NOs: 19 and 20.

Alternatively or additionally, the inventive polypeptide can comprisethe entire length of an α or β chain of one of the TCRs describedherein. In this regard, the inventive polypeptide can comprise an aminoacid sequence of SEQ ID NOs: 23, 24, 25, or 26. Alternatively, thepolypeptide of the invention can comprise α and β chains of the TCRsdescribed herein. For example, the inventive polypeptide can comprisethe amino acid sequences of both SEQ ID NOs: 23 and 24 or the sequencesof both SEQ ID NOs: 25 and 26.

The invention further provides an isolated or purified proteincomprising at least one of the polypeptides described herein. By“protein” is meant a molecule comprising one or more polypeptide chains.

The protein of the invention can comprise a first polypeptide chaincomprising the amino acid sequence of SEQ ID NO: 19 or 35 and a secondpolypeptide chain comprising the amino acid sequence of SEQ ID NO: 20 or36. The protein of the invention can, for example, comprise a firstpolypeptide chain comprising the amino acid sequence of SEQ ID NO: 23 or25 and a second polypeptide chain comprising the amino acid sequence ofSEQ ID NO: 24 or 26. In this instance, the protein of the invention canbe a TCR. Alternatively, if, for example, the protein comprises a singlepolypeptide chain comprising SEQ ID NO: 23 or 25 and SEQ ID NO: 24 or26, or if the first and/or second polypeptide chain(s) of the proteinfurther comprise(s) other amino acid sequences, e.g., an amino acidsequence encoding an immunoglobulin or a portion thereof, then theinventive protein can be a fusion protein. In this regard, the inventionalso provides a fusion protein comprising at least one of the inventivepolypeptides described herein along with at least one other polypeptide.The other polypeptide can exist as a separate polypeptide of the fusionprotein, or can exist as a polypeptide, which is expressed in frame (intandem) with one of the inventive polypeptides described herein. Theother polypeptide can encode any peptidic or proteinaceous molecule, ora portion thereof, including, but not limited to an immunoglobulin, CD3,CD4, CD8, an MHC molecule, a CD1 molecule, e.g., CD1a, CD1b, CD1c, CD1d,etc.

The fusion protein can comprise one or more copies of the inventivepolypeptide and/or one or more copies of the other polypeptide. Forinstance, the fusion protein can comprise 1, 2, 3, 4, 5, or more, copiesof the inventive polypeptide and/or of the other polypeptide. Suitablemethods of making fusion proteins are known in the art, and include, forexample, recombinant methods. See, for instance, Choi et al., Mol.Biotechnol. 31: 193-202 (2005).

In some embodiments of the invention, the TCRs, polypeptides, andproteins of the invention may be expressed as a single proteincomprising a linker peptide linking the α chain and the β chain. In thisregard, the TCRs, polypeptides, and proteins of the invention mayfurther comprise a linker peptide comprising an amino acid sequencecomprising SEQ ID NO: 37. In an embodiment of the invention, the linkerpeptide may be encoded by a nucleotide sequence comprising SEQ ID NO:38. The linker peptide may advantageously facilitate the expression of arecombinant TCR, polypeptide, and/or protein in a host cell. Uponexpression of the construct including the linker peptide by a host cell,the linker peptide may be cleaved, resulting in separated α and βchains.

The protein of the invention can be a recombinant antibody comprising atleast one of the inventive polypeptides described herein. As usedherein, “recombinant antibody” refers to a recombinant (e.g.,genetically engineered) protein comprising at least one of thepolypeptides of the invention and a polypeptide chain of an antibody, ora portion thereof. The polypeptide of an antibody, or portion thereof;can be a heavy chain, a light chain, a variable or constant region of aheavy or light chain, a single chain variable fragment (scFv), or an Fc,Fab, or F(ab)₂′ fragment of an antibody, etc. The polypeptide chain ofan antibody, or portion thereof, can exist as a separate polypeptide ofthe recombinant antibody. Alternatively, the polypeptide chain of anantibody, or portion thereof, can exist as a polypeptide, which isexpressed in frame (in tandem) with the polypeptide of the invention.The polypeptide of an antibody, or portion thereof, can be a polypeptideof any antibody or any antibody fragment, including any of theantibodies and antibody fragments described herein.

Included in the scope of the invention are functional variants of theinventive TCRs, polypeptides, and proteins described herein. The term“functional variant” as used herein refers to a TCR, polypeptide, orprotein having substantial or significant sequence identity orsimilarity to a parent TCR, polypeptide, or protein, which functionalvariant retains the biological activity of the TCR, polypeptide, orprotein of which it is a variant. Functional variants encompass, forexample, those variants of the TCR, polypeptide, or protein describedherein (the parent TCR, polypeptide, or protein) that retain the abilityto specifically bind to SSX-2 for which the parent TCR has antigenicspecificity or to which the parent polypeptide or protein specificallybinds, to a similar extent, the same extent, or to a higher extent, asthe parent TCR, polypeptide, or protein. Alternatively or additionally,functional variants can also encompass, for example, those variants ofthe TCR, polypeptide, or protein described herein (the parent TCR,polypeptide, or protein) that retain the ability to recognize any one ormore of SSX-3, SSX-4, SSX-5, SSX-9, and SSX-10, which the parentpolypeptide or protein recognizes, to a similar extent, the same extent,or to a higher extent, as the parent TCR, polypeptide, or protein. Inreference to the parent TCR, polypeptide, or protein, the functionalvariant can, for instance, be at least about 30%, 50%, 75%, 80%, 90%,95%, 96%, 97%, 98%, 99% or more identical in amino acid sequence to theparent TCR, polypeptide, or protein.

The functional variant can, for example, comprise the amino acidsequence of the parent TCR, polypeptide, or protein with at least oneconservative amino acid substitution. Conservative amino acidsubstitutions are known in the art, and include amino acid substitutionsin which one amino acid having certain physical and/or chemicalproperties is exchanged for another amino acid that has the samechemical or physical properties. For instance, the conservative aminoacid substitution can be an acidic amino acid substituted for anotheracidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar sidechain substituted for another amino acid with a nonpolar side chain(e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basicamino acid substituted for another basic amino acid (Lys, Arg, etc.), anamino acid with a polar side chain substituted for another amino acidwith a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

Alternatively or additionally, the functional variants can comprise theamino acid sequence of the parent TCR, polypeptide, or protein with atleast one non-conservative amino acid substitution. In this case, it ispreferable for the non-conservative amino acid substitution to notinterfere with or inhibit the biological activity of the functionalvariant. Preferably, the non-conservative amino acid substitutionenhances the biological activity of the functional variant, such thatthe biological activity of the functional variant is increased ascompared to the parent TCR, polypeptide, or protein.

The TCR, polypeptide, or protein can consist essentially of thespecified amino acid sequence or sequences described herein, such thatother components of the functional variant, e.g., other amino acids, donot materially change the biological activity of the functional variant.In this regard, the inventive TCR, polypeptide, or protein can, forexample, consist essentially of the amino acid sequence of SEQ ID NO:23, 24, 25, 26, both SEQ ID NOs: 23 and 24, or both SEQ ID NOs: 25 and26. Also, for instance, the inventive TCRs, polypeptides, or proteinscan consist essentially of the amino acid sequence(s) of SEQ ID NO: 19,20, 21, 22, 35, 36, both SEQ ID NOs: 19 and 20, both SEQ ID NOs: 21 and22, or both SEQ ID NOs: 35 and 36. Furthermore, the inventive TCRs,polypeptides, or proteins can consist essentially of the amino acidsequence of SEQ ID NO: 13 (CDR1 of α chain), 14 (CDR2 of α chain), 15(CDR3 of α chain), 16 (CDR1 of β chain), 17 (CDR2 of β chain), 18 (CDR3of β chain), or any combination thereof, e.g., SEQ ID NOs: 13-15, 16-18,or 13-18.

The TCRs, polypeptides, and proteins of the invention (includingfunctional portions and functional variants) can be of any length, i.e.,can comprise any number of amino acids, provided that the TCRs,polypeptides, or proteins (or functional portions or functional variantsthereof) retain their biological activity, e.g., the ability tospecifically bind to SSX-2; recognize any one or more of SSX-3, SSX-4,SSX-5, SSX-9, and SSX-10; detect cancer in a host; or treat or preventcancer in a host, etc. For example, the polypeptide can be 50 to 5000amino acids long, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400,500, 600, 700, 800, 900, 1000 or more amino acids in length. In thisregard, the polypeptides of the invention also include oligopeptides.

The TCRs, polypeptides, and proteins of the invention (includingfunctional portions and functional variants) of the invention cancomprise synthetic amino acids in place of one or morenaturally-occurring amino acids. Such synthetic amino acids are known inthe art, and include, for example, aminocyclohexane carboxylic acid,norleucine, α-amino n-decanoic acid, homoserine,S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline,4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine,4-carboxyphenylalanine, β-phenylserine-β-hydroxyphenylalanine,phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine,indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid, aminomalonic acid, aminomalonic acid monoamide,N′-benzyl-N′-methyl-lysine, N′,N′-dibenzyl-lysine, 6-hydroxylysine,ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptane carboxylic acid,α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid,α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

The TCRs, polypeptides, and proteins of the invention (includingfunctional portions and functional variants) can be glycosylated,amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclizedvia, e.g., a disulfide bridge, or converted into an acid addition saltand/or optionally dimerized or polymerized, or conjugated.

When the TCRs, polypeptides, and proteins of the invention (includingfunctional portions and functional variants) are in the form of a salt,preferably, the polypeptides are in the form of a pharmaceuticallyacceptable salt. Suitable pharmaceutically acceptable acid additionsalts include those derived from mineral acids, such as hydrochloric,hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids,and organic acids, such as tartaric, acetic, citric, malic, lactic,fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids,for example, p-toluenesulphonic acid.

The TCR, polypeptide, and/or protein of the invention (includingfunctional portions and functional variants thereof) can be obtained bymethods known in the art. Suitable methods of de novo synthesizingpolypeptides and proteins are described in references, such as Chan etal., Fmoc Solid Phase Peptide Synthesis, Oxford University Press,Oxford, United Kingdom, 2005; Peptide and Protein Drug Analysis, ed.Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood etal., Oxford University Press, Oxford, United Kingdom, 2000; and U.S.Pat. No. 5,449,752. Also, polypeptides and proteins can be recombinantlyproduced using the nucleic acids described herein using standardrecombinant methods. See, for instance, Sambrook et al., MolecularCloning: A Laboratory Manual, 3^(rd) ed., Cold Spring Harbor Press, ColdSpring Harbor, N.Y. 2001; and Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates and John Wiley & Sons,NY, 1994. Further, some of the TCRs, polypeptides, and proteins of theinvention (including functional portions and functional variantsthereof) can be isolated and/or purified from a source, such as a plant,a bacterium, an insect, a mammal, e.g., a rat, a human, etc. Methods ofisolation and purification are well-known, in the art. Alternatively,the TCRs, polypeptides, and/or proteins described herein (includingfunctional portions and functional variants thereof) can be commerciallysynthesized by companies, such as Synpep (Dublin, Calif.), PeptideTechnologies Corp. (Gaithersburg, Md.), and Multiple Peptide Systems(San Diego, Calif.). In this respect, the inventive TCRs, polypeptides,and proteins can be synthetic, recombinant, isolated, and/or purified.

Included in the scope of the invention are conjugates, e.g.,bioconjugates, comprising any of the inventive TCRs, polypeptides, orproteins (including any of the functional portions or variants thereof),nucleic acids, recombinant expression vectors, host cells, populationsof host cells, or antibodies, or antigen binding portions thereof.Conjugates, as well as methods of synthesizing conjugates in general,are known in the art (See, for instance, Hudecz, F., Methods Mol. Biol.298: 209-223 (2005) and Kirin et al., Inorg Chem. 44(15): 5405-5415(2005)).

Further provided by the invention is a nucleic acid comprising anucleotide sequence encoding any of the TCRs, polypeptides, or proteinsdescribed herein (including functional portions and functional variantsthereof).

By “nucleic acid” as used herein includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means apolymer of DNA or RNA, which can be single-stranded or double-stranded,synthesized or obtained (e.g., isolated and/or purified) from naturalsources, which can contain natural, non-natural or altered nucleotides,and which can contain a natural, non-natural or altered internucleotidelinkage, such as a phosphoroamidate linkage or a phosphorothioatelinkage, instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide. It is generally preferred that thenucleic acid does not comprise any insertions, deletions, inversions,and/or substitutions. However, it may be suitable in some instances, asdiscussed herein, for the nucleic acid to comprise one or moreinsertions, deletions, inversions, and/or substitutions.

Preferably, the nucleic acids of the invention are recombinant. As usedherein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments to nucleic acid molecules that can replicate in a livingcell, or (ii) molecules that result from the replication of thosedescribed in (i) above. For purposes herein, the replication can be invitro replication or in vivo replication.

The nucleic acids can be constructed based on chemical synthesis and/orenzymatic ligation reactions using procedures known in the art. See, forexample, Sambrook et al., supra, and Ausubel et al., supra. For example,a nucleic acid can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed upon hybridization (e.g.,phosphorothioate derivatives and acridine substituted nucleotides).Examples of modified nucleotides that can be used to generate thenucleic acids include, but are not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-substitutedadenine, 7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleicacids of the invention can be purchased from companies, such asMacromolecular Resources (Fort Collins, Colo.) and Synthegen (Houston,Tex.).

The nucleic acid can comprise any nucleotide sequence which encodes anyof the TCRs, polypeptides, or proteins, or functional portions orfunctional variants thereof. For example, the nucleic acid can comprisea nucleotide sequence comprising, consisting of, or consistingessentially of, SEQ ID NO: 27 (encodes anti-SSX-2 TCR alpha and betachains) or SEQ ID NO: 28 (encodes human/mouse chimeric anti-SSX-2 TCRalpha and beta chains). The nucleotide sequence alternatively cancomprise a nucleotide sequence which is degenerate to SEQ ID NO: 27 or28.

In some embodiments, the nucleic acid sequence may be optimized. Withoutbeing bound to a particular theory, it is believed that optimization ofthe nucleic acid sequence increases the translation efficiency of themRNA transcripts. Optimization of the nucleic acid sequence may involvesubstituting a native codon for another codon that encodes the sameamino acid, but can be translated by tRNA that is more readily availablewithin a cell, thus increasing translation efficiency. Optimization ofthe nucleic acid sequence may also reduce secondary mRNA structures thatwould interfere with translation, thus increasing translationefficiency. For example, the optimized nucleic acid can comprise anucleotide sequence comprising, consisting of, or consisting essentiallyof, SEQ ID NO: 29 (encodes anti-SSX-2 TCR alpha and beta chains) or SEQID NO: 30 (encodes human/mouse chimeric anti-SSX-2 TCR alpha and betachains). The nucleotide sequence alternatively can comprise a nucleotidesequence which is degenerate to SEQ ID NO: 29 or 30.

The invention also provides an isolated or purified nucleic acidcomprising a nucleotide sequence which is complementary to thenucleotide sequence of any of the nucleic acids described herein or anucleotide sequence which hybridizes under stringent conditions to thenucleotide sequence of any of the nucleic acids described herein.

The nucleotide sequence which hybridizes under stringent conditionspreferably hybridizes under high stringency conditions. By “highstringency conditions” is meant that the nucleotide sequencespecifically hybridizes to a target sequence (the nucleotide sequence ofany of the nucleic acids described herein) in an amount that isdetectably stronger than non-specific hybridization. High stringencyconditions include conditions which would distinguish a polynucleotidewith an exact complementary sequence, or one containing only a fewscattered mismatches from a random sequence that happened to have a fewsmall regions (e.g., 3-10 bases) that matched the nucleotide sequence.Such small regions of complementarity are more easily melted than afull-length complement of 14-17 or more bases, and high stringencyhybridization makes them easily distinguishable. Relatively highstringency conditions would include, for example, low salt and/or hightemperature conditions, such as provided by about 0.02-0.1 M NaCl or theequivalent, at temperatures of about 50-70° C. Such high stringencyconditions tolerate little, if any, mismatch between the nucleotidesequence and the template or target strand, and are particularlysuitable for detecting expression of any of the inventive TCRs. It isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide.

The nucleic acids of the invention can be incorporated into arecombinant expression vector. In this regard, the invention providesrecombinant expression vectors comprising any of the nucleic acids ofthe invention. For purposes herein, the term “recombinant expressionvector” means a genetically-modified oligonucleotide or polynucleotideconstruct that permits the expression of an mRNA, protein, polypeptide,or peptide by a host cell, when the construct comprises a nucleotidesequence encoding the mRNA, protein, polypeptide, or peptide, and thevector is contacted with the cell under conditions sufficient to havethe mRNA, protein, polypeptide, or peptide expressed within the cell.The vectors of the invention are not naturally-occurring as a whole.However, parts of the vectors can be naturally-occurring. The inventiverecombinant expression vectors can comprise any type of nucleotides,including, but not limited to DNA and RNA, which can be single-strandedor double-stranded, synthesized or obtained in part from naturalsources, and which can contain natural, non-natural or alterednucleotides. The recombinant expression vectors can comprisenaturally-occurring, non-naturally-occurring internucleotide linkages,or both types of linkages. Preferably, the non-naturally occurring oraltered nucleotides or internucleotide linkages does not hinder thetranscription or replication of the vector.

The recombinant expression vector of the invention can be any suitablerecombinant expression vector, and can be used to transform or transfectany suitable host. Suitable vectors include those designed forpropagation and expansion or for expression or both, such as plasmidsand viruses. The vector can be selected from the group consisting of thepUC series (Fermentas Life Sciences), the pBluescript series(Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.),the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series(Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as λGT10,λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, also can be used.Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3,pBI121 and pBIN19 (Clontech). Examples of animal expression vectorsinclude pEUK-C1, pMAM and pMAMneo (Clontech). Preferably, therecombinant expression vector is a viral vector, e.g., a retroviralvector.

The recombinant expression vectors of the invention can be preparedusing standard recombinant DNA techniques described in, for example,Sambrook et al., supra, and Ausubel et al., supra. Constructs ofexpression vectors, which are circular or linear, can be prepared tocontain a replication system functional in a prokaryotic or eukaryotichost cell. Replication systems can be derived, e.g., from ColE1, 2μplasmid, λ, SV40, bovine papilloma virus, and the like.

Desirably, the recombinant expression vector comprises regulatorysequences, such as transcription and translation initiation andtermination codons, which are specific to the type of host (e.g.,bacterium, fungus, plant, or animal) into which the vector is to beintroduced, as appropriate and taking into consideration whether thevector is DNA- or RNA-based.

The recombinant expression vector can include one or more marker genes,which allow for selection of transformed or transfected hosts. Markergenes include biocide resistance, e.g., resistance to antibiotics, heavymetals, etc., complementation in an auxotrophic host to provideprototrophy, and the like. Suitable marker genes for the inventiveexpression vectors include, for instance, neomycin/G418 resistancegenes, hygromycin resistance genes, histidinol resistance genes,tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector can comprise a native or normativepromoter operably linked to the nucleotide sequence encoding the TCR,polypeptide, or protein (including functional portions and functionalvariants thereof), or to the nucleotide sequence which is complementaryto or which hybridizes to the nucleotide sequence encoding the TCR,polypeptide, or protein. The selection of promoters, e.g., strong, weak,inducible, tissue-specific and developmental-specific, is within theordinary skill of the artisan. Similarly, the combining of a nucleotidesequence with a promoter is also within the skill of the artisan. Thepromoter can be a non-viral promoter or a viral promoter, e.g., acytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and apromoter found in the long-terminal repeat of the murine stem cellvirus.

The inventive recombinant expression vectors can be designed for eithertransient expression, for stable expression, or for both. Also, therecombinant expression vectors can be made for constitutive expressionor for inducible expression. Further, the recombinant expression vectorscan be made to include a suicide gene.

As used herein, the term “suicide gene” refers to a gene that causes thecell expressing the suicide gene to die. The suicide gene can be a genethat confers sensitivity to an agent, e.g., a drug, upon the cell inwhich the gene is expressed, and causes the cell to die when the cell iscontacted with or exposed to the agent. Suicide genes are known in theart (see, for example, Suicide Gene Therapy: Methods and Reviews,Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeuticsat the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press,2004) and include, for example, the Herpes Simplex Virus (HSV) thymidinekinase (TK) gene, cytosine daminase, purine nucleoside phosphorylase,and nitroreductase.

Another embodiment of the invention further provides a host cellcomprising any of the recombinant expression vectors described herein.As used herein, the term “host cell” refers to any type of cell that cancontain the inventive recombinant expression vector. The host cell canbe a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be aprokaryotic cell, e.g., bacteria or protozoa. The host cell can be acultured cell or a primary cell, i.e., isolated directly from anorganism, e.g., a human. The host cell can be an adherent cell or asuspended cell, i.e., a cell that grows in suspension. Suitable hostcells are known in the art and include, for instance, DH5αE. coli cells,Chinese hamster ovarian cells, monkey VERO cells, COS cells, HEK293cells, and the like. For purposes of amplifying or replicating therecombinant expression vector, the host cell is preferably a prokaryoticcell, e.g., a DH5α cell. For purposes of producing a recombinant TCR,polypeptide, or protein, the host cell is preferably a mammalian cell.Most preferably, the host cell is a human cell. While the host cell canbe of any cell type, can originate from any type of tissue, and can beof any developmental stage, the host cell preferably is a peripheralblood leukocyte (PBL) or a peripheral blood mononuclear cell (PBMC).More preferably, the host cell is a T cell.

For purposes herein, the T cell can be any T cell, such as a cultured Tcell, e.g., a primary T cell, or a T cell from a cultured T cell line,e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. Ifobtained from a mammal, the T cell can be obtained from numeroussources, including but not limited to blood, bone marrow, lymph node,the thymus, or other tissues or fluids. T cells can also be enriched foror purified. Preferably, the T cell is a human T cell. More preferably,the T cell is a T cell isolated from a human. The T cell can be any typeof T cell and can be of any developmental stage, including but notlimited to, CD4⁺/CD8⁺ double positive T cells, CD4⁺ helper T cells,e.g., Th₁ and Th₂ cells, CD8⁺ T cells (e.g., cytotoxic T cells), tumorinfiltrating cells (TILs), memory T cells, naïve T cells, and the like.Preferably, the T cell is a CD8⁺ T cell or a CD4⁺ T cell.

Also provided by the invention is a population of cells comprising atleast one host cell described herein. The population of cells can be aheterogeneous population comprising the host cell comprising any of therecombinant expression vectors described, in addition to at least oneother cell, e.g., a host cell (e.g., a T cell), which does not compriseany of the recombinant expression vectors, or a cell other than a Tcell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, ahepatocyte, an endothelial cell, an epithelial cells, a muscle cell, abrain cell, etc. Alternatively, the population of cells can be asubstantially homogeneous population, in which the population comprisesmainly of host cells (e.g., consisting essentially of) comprising therecombinant expression vector. The population also can be a clonalpopulation of cells, in which all cells of the population are clones ofa single host cell comprising a recombinant expression vector, such thatall cells of the population comprise the recombinant expression vector.In one embodiment of the invention, the population of cells is a clonalpopulation comprising host cells comprising a recombinant expressionvector as described herein.

The invention further provides an antibody, or antigen binding portionthereof, which specifically binds to a functional portion of any of theTCRs described herein. Preferably, the functional portion specificallybinds to the cancer antigen, e.g., the functional portion comprising theamino acid sequence SEQ ID NO: 13 (CDR1 of α chain), 14 (CDR2 of αchain), 15 (CDR3 of α chain), 16 (CDR1 of β chain), 17 (CDR2 of βchain), 18 (CDR3 of β chain), SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:35, SEQ ID NO: 36, or a combination thereof, e.g., 13-15; 16-18; 13-18;19-20, or 35-36. More preferably, the functional portion comprises theamino acid sequences of SEQ ID NOs: 13-18. In a preferred embodiment,the antibody, or antigen binding portion thereof, binds to an epitopewhich is formed by all 6 CDRs (CDR1-3 of the alpha chain and CDR1-3 ofthe beta chain). The antibody can be any type of immunoglobulin that isknown in the art. For instance, the antibody can be of any isotype,e.g., IgA, IgD, IgE, IgG, IgM, etc. The antibody can be monoclonal orpolyclonal. The antibody can be a naturally-occurring antibody, e.g., anantibody isolated and/or purified from a mammal, e.g., mouse, rabbit,goat, horse, chicken, hamster, human, etc. Alternatively, the antibodycan be a genetically-engineered antibody, e.g., a humanized antibody ora chimeric antibody. The antibody can be in monomeric or polymeric form.Also, the antibody can have any level of affinity or avidity for thefunctional portion of the inventive TCR. Desirably, the antibody isspecific for the functional portion of the inventive TCR, such thatthere is minimal cross-reaction with other peptides or proteins.

Methods of testing antibodies for the ability to bind to any functionalportion of the inventive TCR are known in the art and include anyantibody-antigen binding assay, such as, for example, radioimmunoassay(RIA), ELISA, Western blot, immunoprecipitation, and competitiveinhibition assays (see, e.g., Janeway et al., infra, and U.S. PatentApplication Publication No. 2002/0197266 A1).

Suitable methods of making antibodies are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Köhler andMilstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.),Antibodies: A Laboratory Manual, CSH Press (1988), and C. A. Janeway etal. (eds.), Immunobiology, 5^(th) Ed., Garland Publishing, New York,N.Y. (2001)). Alternatively, other methods, such as EBV-hybridomamethods (Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984),and Roder et al., Methods Enzymol., 121, 140-67 (1986)), andbacteriophage vector expression systems (see, e.g., Huse et al.,Science, 246, 1275-81 (1989)) are known in the art. Further, methods ofproducing antibodies in non-human animals are described in, e.g., U.S.Pat. Nos. 5,545,806, 5,569,825, and 5,714,352, and U.S. PatentApplication Publication No. 2002/0197266 A1).

Phage display furthermore can be used to generate the antibody of theinvention. In this regard, phage libraries encoding antigen-bindingvariable (V) domains of antibodies can be generated using standardmolecular biology, and recombinant DNA techniques (see, e.g., Sambrooket al. (eds.), Molecular Cloning, A Laboratory Manual, 3^(rd) Edition,Cold Spring Harbor Laboratory Press, New York (2001)). Phage encoding avariable region with the desired specificity are selected for specificbinding to the desired antigen, and a complete or partial antibody isreconstituted comprising the selected variable domain. Nucleic acidsequences encoding the reconstituted antibody are introduced into asuitable cell line, such as a myeloma cell used for hybridomaproduction, such that antibodies having the characteristics ofmonoclonal antibodies are secreted by the cell (see, e.g., Janeway etal., supra, Huse et al., supra, and U.S. Pat. No. 6,265,150).

Antibodies can be produced by transgenic mice that are transgenic forspecific heavy and light chain immunoglobulin genes. Such methods areknown in the art and described in, for example U.S. Pat. Nos. 5,545,806and 5,569,825, and Janeway et al., supra.

Methods for generating humanized antibodies are well known in the artand are described in detail in, for example, Janeway et al., supra, U.S.Pat. Nos. 5,225,539, 5,585,089 and 5,693,761, European Patent No.0239400 B1, and United Kingdom Patent No. 2188638. Humanized antibodiescan also be generated using the antibody resurfacing technologydescribed in U.S. Pat. No. 5,639,641 and Pedersen et al., J. Mol. Biol.,235, 959-973 (1994).

The invention also provides antigen binding portions of any of theantibodies described herein. The antigen binding portion can be anyportion that has at least one antigen binding site, such as Fab,F(ab′)₂, dsFv, sFv, diabodies, and triabodies.

A single-chain variable region fragment (sFv) antibody fragment, whichconsists of a truncated Fab fragment comprising the variable (V) domainof an antibody heavy chain linked to a V domain of a light antibodychain via a synthetic peptide, can be generated using routinerecombinant DNA technology techniques (see, e.g., Janeway et al.,supra). Similarly, disulfide-stabilized variable region fragments (dsFv)can be prepared by recombinant DNA technology (see, e.g., Reiter et al.,Protein Engineering, 7, 697-704 (1994)). Antibody fragments of theinvention, however, are not limited to these exemplary types of antibodyfragments.

Also, the antibody, or antigen binding portion thereof, can be modifiedto comprise a detectable label, such as, for instance, a radioisotope, afluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin(PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase),and element particles (e.g., gold particles).

The inventive TCRs, polypeptides, proteins, (including functionalportions and functional variants thereof), nucleic acids, recombinantexpression vectors, host cells (including populations thereof), andantibodies (including antigen binding portions thereof), can be isolatedand/or purified. The term “isolated” as used herein means having beenremoved from its natural environment. The term “purified” as used hereinmeans having been increased in purity, wherein “purity” is a relativeterm, and not to be necessarily construed as absolute purity. Forexample, the purity, can be at least about 50%, can be greater than 60%,70% or 80%, 90% or can be 100%.

The inventive TCRs, polypeptides, proteins (including functionalportions and variants thereof), nucleic acids, recombinant expressionvectors, host cells (including populations thereof), and antibodies(including antigen binding portions thereof), all of which arecollectively referred to as “inventive TCR materials” hereinafter, canbe formulated into a composition, such as a pharmaceutical composition.In this regard, the invention provides a pharmaceutical compositioncomprising any of the TCRs, polypeptides, proteins, functional portions,functional variants, nucleic acids, expression vectors, host cells(including populations thereof), and antibodies (including antigenbinding portions thereof), and a pharmaceutically acceptable carrier.The inventive pharmaceutical compositions containing any of theinventive TCR materials can comprise more than one inventive TCRmaterial, e.g., a polypeptide and a nucleic acid, or two or moredifferent TCRs. Alternatively, the pharmaceutical composition cancomprise an inventive TCR material in combination with anotherpharmaceutically active agents or drugs, such as a chemotherapeuticagents, e.g., asparaginase, busulfan, carboplatin, cisplatin,daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea,methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.

Preferably, the carrier is a pharmaceutically acceptable carrier. Withrespect to pharmaceutical compositions, the carrier can be any of thoseconventionally used and is limited only by chemico-physicalconsiderations, such as solubility and lack of reactivity with theactive compound(s), and by the route of administration. Thepharmaceutically acceptable carriers described herein, for example,vehicles, adjuvants, excipients, and diluents, are well-known to thoseskilled in the art and are readily available to the public. It ispreferred that the pharmaceutically acceptable carrier be one which ischemically inert to the active agent(s) and one which has no detrimentalside effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particularinventive TCR material, as well as by the particular method used toadminister the inventive TCR material. Accordingly, there are a varietyof suitable formulations of the pharmaceutical composition of theinvention. The following formulations for oral, aerosol, parenteral,subcutaneous, intravenous, intramuscular, intraarterial, intrathecal,and interperitoneal administration are exemplary and are in no waylimiting. More than one route can be used to administer the inventiveTCR materials, and in certain instances, a particular route can providea more immediate and more effective response than another route.

Topical formulations are well-known to those of skill in the art. Suchformulations are particularly suitable in the context of the inventionfor application to the skin.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the inventive TCR materialdissolved in diluents, such as water, saline, or orange juice; (b)capsules, sachets, tablets, lozenges, and troches, each containing apredetermined amount of the active ingredient, as solids or granules;(c) powders; (d) suspensions in an appropriate liquid; and (e) suitableemulsions. Liquid formulations may include diluents, such as water andalcohols, for example, ethanol, benzyl alcohol, and the polyethylenealcohols, either with or without the addition of a pharmaceuticallyacceptable surfactant. Capsule forms can be of the ordinary hard- orsoft-shelled gelatin type containing, for example, surfactants,lubricants, and inert fillers, such as lactose, sucrose, calciumphosphate, and corn starch. Tablet forms can include one or more oflactose, sucrose, mannitol, corn starch, potato starch, alginic acid,microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicondioxide, croscarmellose sodium, talc, magnesium stearate, calciumstearate, zinc stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, disintegrating agents, moistening agents,preservatives, flavoring agents, and other pharmacologically compatibleexcipients. Lozenge forms can comprise the inventive TCR material in aflavor, usually sucrose and acacia or tragacanth, as well as pastillescomprising the inventive TCR material in an inert base, such as gelatinand glycerin, or sucrose and acacia, emulsions, gels, and the likecontaining, in addition to, such excipients as are known in the art.

The inventive TCR material, alone or in combination with other suitablecomponents, can be made into aerosol formulations to be administered viainhalation. These aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like. They also may be formulated as pharmaceuticalsfor non-pressured preparations, such as in a nebulizer or an atomizer.Such spray formulations also may be used to spray mucosa.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The inventive TCR material can be administered in a physiologicallyacceptable diluent in a pharmaceutical carrier, such as a sterile liquidor mixture of liquids, including water, saline, aqueous dextrose andrelated sugar solutions, an alcohol, such as ethanol or hexadecylalcohol, a glycol, such as propylene glycol or polyethylene glycol,dimethylsulfoxide, glycerol, ketals such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400,oils, fatty acids, fatty acid esters or glycerides, or acetylated fattyacid glycerides with or without the addition of a pharmaceuticallyacceptable surfactant, such as a soap or a detergent, suspending agent,such as pectin, carbomers, methylcellulose,hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifyingagents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-β-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations will typically contain from about 0.5% toabout 25% by weight of the inventive TCR material in solution.Preservatives and buffers may be used. In order to minimize or eliminateirritation at the site of injection, such compositions may contain oneor more nonionic surfactants having a hydrophile-lipophile balance (HLB)of from about 12 to about 17. The quantity of surfactant in suchformulations will typically range from about 5% to about 15% by weight.Suitable surfactants include polyethylene glycol sorbitan fatty acidesters, such as sorbitan monooleate and the high molecular weightadducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol. The parenteralformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

Injectable formulations are in accordance with the invention. Therequirements for effective pharmaceutical carriers for injectablecompositions are well-known to those of ordinary skill in the art (see,e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630(1986)). Preferably, when administering cells, e.g., T cells, the cellsare administered via injection.

It will be appreciated by one of skill in the art that, in addition tothe above-described pharmaceutical compositions, the inventive. TCRmaterials of the invention can be formulated as inclusion complexes,such as cyclodextrin inclusion complexes, or liposomes.

For purposes of the invention, the amount or dose of the inventive TCRmaterial administered should be sufficient to effect, e.g., atherapeutic or prophylactic response, in the subject or animal over areasonable time frame. For example, the dose of the inventive TCRmaterial should be sufficient to bind to a cancer antigen, or detect,treat or prevent cancer in a period of from about 2 hours or longer,e.g., 12 to 24 or more hours, from the time of administration. Incertain embodiments, the time period could be even longer. The dose willbe determined by the efficacy of the particular inventive TCR materialand the condition of the animal (e.g., human), as well as the bodyweight of the animal (e.g., human) to be treated.

Many assays for determining an administered dose are known in the art.For purposes of the invention, an assay, which comprises comparing theextent to which target cells are lysed or IFN-γ is secreted by T cellsexpressing the inventive TCR, polypeptide, or protein uponadministration of a given dose of such T cells to a mammal among a setof mammals of which is each given a different dose of the T cells, couldbe used to determine a starting dose to be administered to a mammal. Theextent to which target cells are lysed or IFN-γ is secreted uponadministration of a certain dose can be assayed by methods known in theart.

The dose of the inventive TCR material also will be determined by theexistence, nature and extent of any adverse side effects that mightaccompany the administration of a particular inventive TCR material.Typically, the attending physician will decide the dosage of theinventive TCR material with which to treat each individual patient,taking into consideration a variety of factors, such as age, bodyweight, general health, diet, sex, inventive TCR material to beadministered, route of administration, and the severity of the conditionbeing treated. By way of example and not intending to limit theinvention, the dose of the inventive TCR material can be about 0.001 toabout 1000 mg/kg body weight of the subject being treated/day, fromabout 0.01 to about 10 mg/kg body weight/day, about 0.01 mg to about 1mg/kg body weight/day.

One of ordinary skill in the art will readily appreciate that theinventive TCR materials of the invention can be modified in any numberof ways, such that the therapeutic or prophylactic efficacy of theinventive TCR materials is increased through the modification. Forinstance, the inventive TCR materials can be conjugated either directlyor indirectly through a bridge to a targeting moiety. The practice ofconjugating compounds, e.g., inventive TCR materials, to targetingmoieties is known in the art. See, for instance, Wadwa et al., J. DrugTargeting 3: 111 (1995) and U.S. Pat. No. 5,087,616. The term “targetingmoiety” as used herein, refers to any molecule or agent thatspecifically recognizes and binds to a cell-surface receptor, such thatthe targeting moiety directs the delivery of the inventive TCR materialsto a population of cells on which surface the receptor is expressed.Targeting moieties include, but are not limited to, antibodies, orfragments thereof, peptides, hormones, growth factors, cytokines, andany other natural or non-natural ligands, which bind to cell surfacereceptors (e.g., Epithelial Growth Factor Receptor (EGFR), T-cellreceptor (TCR), B-cell receptor (BCR), CD28, Platelet-derived GrowthFactor Receptor (PDGF), nicotinic acetylcholine receptor (nAChR), etc.).The term “bridge” as used herein, refers to any agent or molecule thatlinks the inventive TCR materials to the targeting moiety. One ofordinary skill in the art recognizes that sites on the inventive TCRmaterials, which are not necessary for the function of the inventive TCRmaterials, are ideal sites for attaching a bridge and/or a targetingmoiety, provided that the bridge and/or targeting moiety, once attachedto the inventive TCR materials, do(es) not interfere with the functionof the inventive TCR materials, i.e., the ability to bind to SSX-2;recognize any one or more of SSX-3, SSX-4, SSX-5, SSX-9, and SSX-10; orto detect, treat, or prevent cancer.

Alternatively, the inventive TCR materials can be modified into a depotform, such that the manner in which the inventive TCR materials isreleased into the body to which it is administered is controlled withrespect to time and location within the body (see, for example, U.S.Pat. No. 4,450,150). Depot forms of inventive TCR materials can be, forexample, an implantable composition comprising the inventive TCRmaterials and a porous or non-porous material, such as a polymer,wherein the inventive TCR materials is encapsulated by or diffusedthroughout the material and/or degradation of the non-porous material.The depot is then implanted into the desired location within the bodyand the inventive TCR materials are released from the implant at apredetermined rate.

In an embodiment of the invention, the pharmaceutical compositionfurther comprises 5-aza-2′-deoxycytidine (DAC). Without being bound to aparticular theory, it is believed that the demethylating agent DACenhances the recognition of cancer cells by any of the inventive TCRmaterials by upregulating expression of SSX-2 by cancer cells.

It is contemplated that the inventive pharmaceutical compositions, TCRs,polypeptides, proteins, nucleic acids, recombinant expression vectors,antibodies, host cells, or populations of cells can be used in methodsof treating or preventing cancer. Without being bound to a particulartheory, the inventive TCRs are believed to bind specifically to SSX-2and may also recognize any one or more of SSX-3, SSX-4, SSX-5, SSX-9,and SSX-10, such that the TCR (or related inventive polypeptide orprotein) when expressed by a cell is able to mediate an immune responseagainst the cell expressing SSX-2 and may also mediate an immuneresponse against any one or more of SSX-3, SSX-4, SSX-5, SSX-9, andSSX-10. In this regard, the invention provides a method of treating orpreventing cancer in a host, comprising administering to the host any ofthe pharmaceutical compositions, TCRs, polypeptides, or proteinsdescribed herein, any nucleic acid or recombinant expression vectorcomprising a nucleotide sequence encoding any of the TCRs, polypeptides,proteins described herein, any of the antibodies described herein, orany host cell or population of cells comprising a recombinant vectorwhich encodes any of the TCRs, polypeptides, or proteins describedherein, in an amount effective to treat or prevent cancer in the host.

In an embodiment of the invention, the method of treating or preventingcancer in a host further comprises administering DAC to the host. Themethod may comprise administering DAC prior to, concurrently with, orafter administering any of the inventive pharmaceutical compositions,TCRs, polypeptides, proteins, nucleic acids, recombinant expressionvectors, host cells, or populations of cells to the host. Without beingbound to a particular theory, it is believed that the demethylatingagent DAC enhances the recognition of cancer cells by any of theinventive TCR materials by upregulating expression of SSX-2 by cancercells.

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof cancer in a mammal. Furthermore, the treatment or prevention providedby the inventive method can include treatment or prevention of one ormore conditions or symptoms of the disease, e.g., cancer, being treatedor prevented. Also, for purposes herein, “prevention” can encompassdelaying the onset of the disease, or a symptom or condition thereof.

Also provided is a method of detecting the presence of cancer in a host.The method comprises (i) contacting a sample comprising cells of thecancer any of the inventive TCRs, polypeptides, proteins, nucleic acids,recombinant expression vectors, host cells, populations of cells, orantibodies, or antigen binding portions thereof, described herein,thereby forming a complex, and detecting the complex, wherein detectionof the complex is indicative of the presence of cancer in the host.

With respect to the inventive method of detecting cancer in a host, thesample of cells of the cancer can be a sample comprising whole cells,lysates thereof, or a fraction of the whole cell lysates, e.g., anuclear or cytoplasmic fraction, a whole protein fraction, or a nucleicacid fraction.

For purposes of the inventive detecting method, the contacting step cantake place in vitro or in vivo with respect to the host. Preferably, thecontacting is in vitro.

Also, detection of the complex can occur through any number of waysknown in the art. For instance, the inventive TCRs, polypeptides,proteins, nucleic acids, recombinant expression vectors, host cells,populations of cells, or antibodies, or antigen binding portionsthereof, described herein, can be labeled with a detectable label suchas, for instance, a radioisotope, a fluorophore (e.g., fluoresceinisothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkalinephosphatase, horseradish peroxidase), and element particles (e.g., goldparticles).

For purposes of the inventive methods, wherein host cells or populationsof cells are administered, the cells can be cells that are allogeneic orautologous to the host. Preferably, the cells are autologous to thehost.

With respect to the inventive methods, the cancer can be any cancer,including any of sarcomas (e.g., synovial sarcoma, osteogenic sarcoma,leiomyosarcoma uteri, and alveolar rhabdomyosarcoma), lymphomas (e.g.,Hodgkin lymphoma and non-Hodgkin lymphoma), hepatocellular carcinoma,glioma, head-neck cancer, acute lymphocytic cancer, acute myeloidleukemia, bone cancer, brain cancer, breast cancer, cancer of the anus,anal canal, or anorectum, cancer of the eye, cancer of the intrahepaticbile duct, cancer of the joints, cancer of the neck, gallbladder, orpleura, cancer of the nose, nasal cavity, or middle ear, cancer of theoral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronicmyeloid cancer, colon cancer (e.g., colon carcinoma), esophageal cancer,cervical cancer, gastrointestinal carcinoid tumor, hypopharynx cancer,larynx cancer, liver cancer, lung cancer, malignant mesothelioma,melanoma, multiple myeloma, nasopharynx cancer, ovarian cancer,pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynxcancer, prostate cancer, rectal cancer, renal cancer, small intestinecancer, soft tissue cancer, stomach cancer, testicular cancer, thyroidcancer, ureter cancer, and urinary bladder cancer. Of these, sarcomas(e.g., synovial sarcoma, osteogenic sarcoma, leiomyosarcoma uteri, andalveolar rhabdomyosarcoma), hepatocellular carcinoma, glioma, livercancer, melanoma, ovarian cancer, pancreatic cancer, and prostate cancerare preferably treated.

An embodiment of the invention provides the use of any of the TCRs,polypeptides, proteins, nucleic acids, recombinant expression vectors,host cells, populations of cells, antibodies or antigen binding portionsthereof, or pharmaceutical compositions, for the treatment or preventionof cancer in a host. In an embodiment, the use may further comprise theuse of DAC.

The host referred to in the inventive methods can be any host.Preferably, the host is a mammal. As used herein, the term “mammal”refers to any mammal, including, but not limited to, mammals of theorder Rodentia, such as mice and hamsters, and mammals of the orderLogomorpha, such as rabbits. It is preferred that the mammals are fromthe order Carnivora, including Felines (cats) and Canines (dogs). It ismore preferred that the mammals are from the order Artiodactyla,including Bovines (cows) and Swines (pigs) or of the orderPerssodactyla, including Equines (horses). It is most preferred that themammals are of the order Primates, Ceboids, or Simoids (monkeys) or ofthe order Anthropoids (humans and apes). An especially preferred mammalis the human.

The following examples further illustrate the invention but should notbe construed as in any way limiting its scope.

EXAMPLE 1

This example illustrates the construction of a retroviral vector forexpressing SSX-2 and demonstrates SSX-2 expression in certain celllines.

The SSX-2 gene was inserted into expression vectors pMSGV1 andpRRLSIN.cPPT.PGK. The sequence of SSX-2 inserted was:

(SEQ ID NO: 31)ATGaacggagacgacgcctttgcaaggagacccacggttggtgctcaaataccagagaagatccaaaaggccttcgatgatattgccaaatacttctctaaggaagagtgggaaaagatgaaagcctcggagaaaatcttctatgtgtatatgaagagaaagtatgaggctatgactaaactaggtttcaaggccaccctcccacctttcatgtgtaataaacgggccgaagacttccaggggaatgatttggataatgaccctaaccgtgggaatcaggttgaacgtcctcagatgactttcggcaggctccagggaatctccccgaagatcatgcccaagaagccagcagaggaaggaaatgattcggaggaagtgccagaagcatctggcccacaaaatgatgggaaagagctgtgccccccgggaaaaccaactacctctgagaagattcacgagagatctggaaatagggaggcccaagaaaaggaagagagacgcggaacagctcatcggtggagcagtcagaacacacacaacattggtcgattcagtttgtcaacttctatgggtgcagttcatggtacccccaaaacaattacacacaacagggacccaaaaggggggaacatgcctggacccacagactgcgtgagagaaaacagaggTGA.

The pRRLSIN vector also had WPRE (Woodchuck Hepatitis VirusPosttranscriptional Regulatory Element) inserted.

Expression of SSX-2 was observed by Western blot in 624.38 cells andalso in COS7-A2 cells that were transduced with an SSX2 vector as above.No SSX-2 expression was observed in H508, Panc2551, A549, or OVCAR3cells or non-transduced COS7-A2 cells.

SSX-2 was also measured in 938mel, U251, T567A, SKMEL23, and SKMEL37cells. The copy number of SSX-2 normalized to β-actin is shown in Table1.

TABLE 1 Cell Line Copy Number SSX-2/10⁶ β-actin 938mel 17194.1 U2516168.2 T567A 8278.0 SKMEL23 0.2 SKMEL37 26568.9

Additional SSX-2 expression studies were performed using real time PCR.The results are shown in Table 2

TABLE 2 Copy Number SSX-2/ Tumor cell line Histology 10⁶ β-actin Capan1Pancreatic cancer 1447027 CRL1837 Pancreatic cancer 10 Panc1 Pancreaticcancer 255 BxPC3 Pancreatic cancer 1653 Panc2551 Pancreatic cancer 32641SW1990 Pancreatic cancer 27 MiaPaca2 Pancreatic cancer 212 HPAF-IIPancreatic cancer 0 H766T Pancreatic cancer 0 HPAC Pancreatic cancer 0H508 Colon cancer 5786 HCT116 Colon cancer 0 SW620 Colon cancer 0 A549Lung cancer 153 H2087 Lung cancer 54 H1299/A2 Lung cancer 194 H2126/A2Lung cancer 204 H446 Lung cancer 550 H596 Lung cancer 88 H2066 Lungcancer 80 H2122 Lung cancer 0 SKLC17 Lung cancer 1696 H82 Lung cancer2303 CALU6 Lung cancer 0 H522 Lung cancer 1 H358 Lung cancer 0 H446 Lungcancer 4 H1688 Lung cancer nd H157 Lung cancer 0 H1250 Lung cancer 266H2721 Lung cancer 75 OvCar3 Ovarian cancer 854 SKOV3 Ovarian cancer 739MDA-MB-231 Breast cancer 0 MDA468 Breast cancer 0 MCF7 Breast cancer 2701300 Melanoma 57 1359 Melanoma 1391 586 Melanoma 327 888 Melanoma 137624.38 Melanoma 915 2984 Melanoma 111 526-NY-ESO Melanoma 0 SKMEL23Melanoma 1 SKMEL37 Melanoma 18833 T567A Melanoma 7426 T331A Melanoma 4[redacted name Renal cancer 193 of cell donor] Toledo Lymphoma 166 NALM6Leukemia 0 U251 Glioma 25642 397/A2 10782 SK-N-AS Neuroblastoma 1 PBLNormal 36 lymphocytes 293GP 0 293-SSX2/A2+ 478175 COST 0 COS-SSX2/A2+616981

EXAMPLE 2

This example illustrates the construction of a retroviral vector forexpressing an SSX-2 specific TCR.

An HLA-A2 restricted TCR from a natural T cell clone was isolated using5′-RACE from a tumor-infiltrated lymph node (TILN) from a melanomapatient seropositive for SSX-2 and whose tumor expressed SSX-2.

The T cell clone showed: TRAV14/DV4*01 (number of bacterial clonespositive 21/23) and TRBV15*02-CB1 (number of bacterial clones positive23/23).

A retroviral vector was constructed expressing the TCR α and β chainsincorporating the 2a cleavage peptide. Separate PCR reactions for the αchain and the β chain were performed. For the α chain, the forwardprimer incorporated an NcoI restriction site of ATG. The reverse primerhad furin-SGSG-P2a incorporated before the recognition sequence. For theβ chain, the forward primer also incorporated furin-SGSG-P2a before therecognition sequence, and the reverse primer had a stop codon and NotIrestriction site.

Upon completion, the separate PCR reactions were combined and additionalPCR performed with outside primers to generate an α chain(TRAV14)-linker-β chain (TRBV15-CB1) construct. The construct containedSEQ ID NO: 27, encoding anti-SSX-2 TCR alpha and beta chains.

The construct was cloned into the pMSGV1 retroviral vector using theNcoI and NotI restriction sites.

EXAMPLE 3

This example demonstrates that PBLs engineered with a SSX-2 TCR showSSX-2 peptide specific reactivity and tetramer binding.

Human donor-derived PBLs were transduced with the SSX-2 TCR vector ofExample 2 and tested for peptide reactivity and tetramer binding.

Tetramer binding was observed in CD4 and CD8 cells.

SSX-2 TCR-transduced PBLs were co-cultured with T2 cells from two humandonors, where the T2 cells were pulsed with varying concentrations ofthe SSX-2: 41-49 (KASEKIFYV) peptide. FIGS. 1A and 1B show the resultinginterferon-γ levels (pg/ml) measured. These data show that SSX-2TCR-transduced PBLs recognize SSX-2: 41-49 peptide down to 0.01 ng/ml,or less. The differences between FIGS. 1A and 1B may be due to donorvariability.

SSX-2 TCR-transduced PBLs were also co-cultured with cells from Example1 retrovirally engineered to express the SSX-2 gene. FIG. 2 shows theresulting interferon-γ levels (pg/ml) measured when the PBLs wereco-cultured with 293-A2 and COST-A2 cells expressing the SSX-2 gene andT2 cells pulsed with the SSX-2: 41-49 peptide. PBLs that were nottransduced with a SSX-2 TCR showed no reactivity against these cells.

EXAMPLE 4

This example demonstrates that PBLs engineered with the SSX-2 TCR ofExample 2 show reactivity against tumor cell lines.

SSX-2 TCR-transduced PBLs were co-cultured with various tumor celllines. FIG. 3 shows the resulting interferon-γ levels (pg/ml) measured.These data show that SSX-2 TCR engineered T cells recognized naturallyprocessed and presented SSX-2 protein in both melanoma (624, and 1300)and glioblastoma cell lines (U251). PBLs that were not transduced with aSSX-2 TCR (UT) showed very little or no reactivity.

FIGS. 4A and 4B, FIG. 11, and Tables 3 and 4 show additional results ofPBLs from human donors that were transduced with the SSX-2 TCR ofExample 2 when these PBLs were co-cultured with various tumor cells.

TABLE 3 UT SSX2-TCR IFN-γ IFN-γ Cell line Histology (pg/ml) (pg/ml)COS-A2 144 297 293-A2 25 173 888 45 126 OVCAR3 0 0 MCF7 0 0 SKMEL 23 0 0T331A 0 0 COS-A2 SSX2 71 36070 624 Melanoma 0 26515 938-A2 Melanoma 019320 938 0 0 293-A2 SSX2 0 33913 U251 Glioma 0 9770 SK MEL37 Melanoma188 10000 SKOV3 Ovarian 0 663 H82 0 141 HEPG2 0 25 T567A Melanoma 15210280 MEDIUM 0 0

TABLE 4 UT SSX2-TCR IFN-γ IFN-γ Cell line Histology (pg/ml) (pg/ml)COS-A2 280 180 293-A2 112 144 888 115 143 OVCAR3 8 21 MCF7 74 3 SKMEL 236 0 T331A 122 42 COS-A2 SSX2 123 56820 624 Melanoma 0 21730 938-A2Melanoma 38 19192 938 0 0 293-A2 SSX2 125 20595 U251 Glioma 0 12025 SKMEL37 Melanoma 313 9720 SKOV3 Ovarian 93 610 H82 0 100 HEPG2 73 61 T567AMelanoma 382 13245 MEDIUM 25 0

EXAMPLE 5

This example demonstrates that PBLs engineered with a SSX-2 TCR showreactivity against other SSX protein peptides.

Co-culture assays were performed with PBLs transduced with the SSX-2 TCRof Example 2 and peptide-pulsed T2 cells.

FIG. 5 shows the SSX-2 TCR is most reactive with the peptide of SSX-2and also recognizes the peptides of SSX-3, -4, -5, -9, and -10 over theother SSX peptides, although greater recognition is seen at higherpeptide concentrations.

EXAMPLE 6

This example demonstrates that codon optimization and introduction of amouse constant region improved the expression and function of SSX-2 TCRin human PBLs.

Human PBL were untransduced or transduced with a vector comprising SEQID NO: 27 (SSX-2 TCR), SEQ ID NO: 29 (codon-optimized SSX-2 TCR), or SEQID NO: 30 (codon-optimized SSX-2 TCR including mouse constant region).Expression was measured by FACS analysis. Mean fluorescence intensitywas measured to be 656 for cells transduced with SEQ ID NO: 27 (SSX-2TCR), 910 for cells transduced with SEQ ID NO: 29 (codon-optimized SSX-2TCR), and 949 for cells transduced with SEQ ID NO: 30 (codon-optimizedSSX-2 TCR including mouse constant region).

In another experiment, measurement of tetramer binding confirmed thatcodon optimization and introduction of a mouse constant region improvedthe expression of SSX-2 TCR in human PBLs.

In another experiment, FACS analysis also revealed that the activationmarker CD137 (4-1BB) was upregulated following co-culture of human PBLtransduced with SEQ ID NO: 27 (SSX-2 TCR), SEQ ID NO: 29(codon-optimized SSX-2 TCR), or SEQ ID NO: 30 (codon-optimized SSX-2 TCRincluding mouse constant region) with COS-A2-SSX2 cells.

Another experiment evaluated the function of the SSX-2 TCRs by measuringCD107a mobilization following coculture with COS-A2-SSX-2 cells. On Day0, PBL were stimulated with OKT3. On Day 2, the PBL were transduced asdescribed in this example. On Day 15, the PBL were co-cultured for 2hours with COS-A2 cells or COS-A2-SSX-2 cells. On Day 16, CD107amobilization was measured by FACS analysis. The results showed thatcodon optimization and introduction of a mouse constant region improvedthe function of SSX-2 TCR as measured by CD107a mobilization followingco-culture with COS-A2-SSX-2 cells.

The function of the SSX-2 TCRs was also measured by IL-2 and IFN-γproduction following co-culture with COS-A2-SSX-2 cells or 938-A2 melcells. PBL were stimulated with OKT3 and transduced as described in thisexample. On Day 10, the PBL were co-cultured with COS-A2 cells,COS-A2-SSX-2 cells, 938-A2 mel cells, or 938mel cells. On Day 11, IL-2and IFN-γ production was measured by FACS analysis. The results showedthat codon optimization and introduction of a mouse constant regionimproved the function of SSX-2 TCR as measured by IL-2 and IFN-γproduction following co-culture with COS-A2-SSX-2 cells and 938-A2 melcells.

EXAMPLE 7

This example demonstrates that PBLs engineered with the SSX-2 TCR,codon-optimized SSX-2 TCR, or a codon-optimized human-mouse chimeraSSX-2 TCR show reactivity against tumor cell lines.

PBLs that were untransduced (UT) or transduced with a vector comprisingSEQ ID NO: 27 (SSX-2 TCR), SEQ ID NO: 29 (codon-optimized SSX-2 TCR), orSEQ ID NO: 30 (codon-optimized SSX-2 TCR including mouse constantregion) were co-cultured with various tumor cell lines. Tables 5 and 6show the resulting interferon-γ levels (pg/ml) measured with respect toPBL from two different donors. These data show that the transduced Tcells recognized naturally processed and presented SSX-2 protein inmultiple tumor cell lines. PBLs that were not transduced with a SSX-2TCR (UT) showed very little or no reactivity.

TABLE 5 SSX2- SSX2- SSX2-TCR-WT TCR-Co TCR-MCR (SEQ ID NO: Op (SEQ ID(SEQ ID Cell line UT 27) NO: 29) NO: 30) T cell alone 445 165 164 327K562 1950 1969 1714 2752 Lau 149 mel 925 285 193 270 T331A mel 47 30 3030 Cos-A2 1280 2839 2338 2827 293-A2 1234 582 711 802 938 mel 1117 1239890 1122 Cos-A2-SSX2 615 52577 64142 56893 293-A2-SSX2 515 29804 3752237258 K562-A2- 1830 12542 21325 17437 Erythroleukemia Skmel 37 mel 966635 8869 10401 1300 mel 176 2556 2596 2715 624 mel 453 27344 3754746999 938-A2 mel 626 37032 46304 51092 U251 Glioma 372 16653 19223 19027SKOV3 Ovarian 877 2414 2527 2221

TABLE 6 SSX2- SSX2-TCR-Co SSX2-TCR- Cell line UT TCR-WT Op MCR T cellalone 180 322 290 554 K562 1734 1807 2784 3328 Lau 149 mel 124 125 120142 T331A mel 115 58 30 38 Cos-A2 252 460 601 553 293-A2 994 1520 10671005 938 mel 915 1451 768 932 Cos-A2-SSX2 65 85892 138324 164314293-A2-SSX2 1027 52481 49112 47273 K562-A2- 1945 12825 13610 13555Erythroleukemia Skmel 37 232 8941 11445 10630 1300 mel 258 3162 30523460 624 mel 2340 60174 47059 57693 938-A2 mel 2175 46094 51173 40047U251 Glioma 656 22888 21418 20027 SKOV3 Ovarian 652 10953 22509 9857

EXAMPLE 8

This example demonstrates that PBLs engineered with the SSX-2 TCR,codon-optimized SSX-2 TCR, or a codon-optimized human-mouse chimeraSSX-2 TCR proliferate upon co-culture with SSX2+/HLA-A2+ target cells.

PBLs from Donor 1 or Donor 2 that were untransduced (UT) or transducedwith a vector comprising SEQ ID NO: 27 (SSX-2 TCR), SEQ ID NO: 29(codon-optimized SSX-2 TCR), or SEQ ID NO: 30 (codon-optimized SSX-2 TCRincluding mouse constant region) were co-cultured with COS-A2-SSX-2cells. Proliferation in terms of [³H]-thymidine incorporation (CPM) wasmeasured and is shown in FIGS. 6A (Donor 1) and 6B (Donor 2). These datashow that PBLs transduced with SSX-2 TCR, codon-optimized SSX-2 TCR, orcodon-optimized SSX-2 TCR including mouse constant region proliferate inresponse to co-culture with COS-A2-SSX-2 cells.

In another experiment, PBLs transduced as described in this example wereco-cultured with 1300 mel cells, 624 mel cells, 888 mel cells, SK mel 37cells, or COS-A2-SSX-2 cells. Proliferation in terms of [³H]-thymidineincorporation counts per minute (CPM) was measured and is shown in FIGS.9A-9E. These data show that PBLs transduced with SSX-2 TCR,codon-optimized SSX-2 TCR, or codon-optimized SSX-2 TCR including mouseconstant region proliferate in response to co-culture with SSX2+/HLA-A2+target cells.

EXAMPLE 9

This example demonstrates that PBLs engineered with the SSX-2 TCR,codon-optimized SSX-2 TCR, or a codon-optimized human-mouse chimeraSSX-2 TCR show specific lytic activity against SSX-2⁺/HLA-A2⁺ targetcells.

PBLs that were untransduced (UT) or transduced with a vector comprisingSEQ ID NO: 27 (SSX-2 TCR), SEQ ID NO: 29 (codon-optimized SSX-2 TCR), orSEQ ID NO: 30 (codon-optimized SSX-2 TCR including mouse constantregion) were co-cultured with target cells 938 mel (HLA-A2−/SSX-2+),COS-A2, 938-A2 mel, COS-A2-SSX-2, 624 mel, 1300 mel, SK mel 37, or 888mel at the effector to target ratios set forth in FIGS. 7A-D and 8A-D.Percent lysis of the target cells was measured and is shown in FIGS.7A-D and 8A-D. Untransduced cells showed little to no reactivity. Thesedata show that PBLs engineered with the SSX-2 TCR, codon-optimized SSX-2TCR, or a codon-optimized human-mouse chimera SSX-2 TCR show specificlytic activity against SSX-2⁺/HLA-A2⁺ target cells.

EXAMPLE 10

This example demonstrates that PBLs engineered with the SSX-2 TCR,codon-optimized SSX-2 TCR, or a codon-optimized human-mouse chimeraSSX-2 TCR secrete cytokine when co-cultured with peptide-pulsed T2cells.

PBLs that were untransduced (UT) or transduced with a vector comprisingSEQ ID NO: 27 (SSX-2 TCR), SEQ ID NO: 29 (codon optimized SSX-2 TCR), orSEQ ID NO: 30 (codon-optimized SSX-2 TCR including mouse constantregion) were co-cultured with T2 cells that were pulsed with an SSX-2were pulsed with varying concentrations of the SSX-2: 41-49 (KASEKIFYV)(SEQ ID NO: 1) peptide. FIG. 10 shows the resulting interferon-γ levels(pg/ml) measured. These data show that SSX-2 TCR-transduced PBLsrecognize SSX-2: 41-49 peptide.

EXAMPLE 11

This example demonstrates that the demethylating agent,5-aza-2′-deoxycytidine (DAC), enhances the recognition of mel1300 cellsby SSX2-TCR engineered PBL.

SSX-2 TCR-transduced PBLs from three donors were co-cultured withmel1300 cells without DAC or with 0.1 μM or 1.0 μM DAC. FIG. 12 showsthe resulting interferon-γ levels (pg/ml) measured. These data show thatDAC enhances the recognition of mel1300 cells by SSX2-TCR engineeredPBL.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. An isolated or purified T cell receptor(TCR) comprising the amino acid sequences of SEQ ID NOs: 13-18, SEQ IDNO: 21 and SEQ ID NO: 22, wherein the TCR has antigenic specificity forsynovial sarcoma X Breakpoint (SSX)-2 (SEQ ID NO: 1) presented in thecontext of an HLA-A2 molecule, wherein the TCR also recognizes SSX-4(SEQ ID NO: 4), and wherein the TCR is a human/mouse chimeric TCR. 2.The TCR of claim 1, wherein the TCR also recognizes any one or more ofSSX-3 (SEQ ID NO: 3), SSX-5 (SEQ ID NO: 5), SSX-9 (SEQ ID NO: 6), andSSX-10 (SEQ ID NO: 7).
 3. The TCR of claim 1, wherein the TCR hasantigenic specificity for an SSX-2 peptide comprising KASEKIFYV (SEQ IDNO: 2).
 4. The TCR of claim 1, wherein the TCR recognizes any one ormore of KVSEKIVYV (SEQ ID NO: 8), KSSEKIVYV (SEQ ID NO: 9), KASEKHYV(SEQ ID NO: 10), KSSEKIIYV (SEQ ID NO: 11), and KASEKILYV (SEQ ID NO:12).
 5. The TCR of claim 1, wherein the TCR comprises SEQ ID NOs: 19 and20.
 6. The TCR of claim 1, wherein the TCR comprises SEQ ID NO: 25 and26.
 7. An isolated or purified polypeptide comprising a functionalportion of the TCR of claim 1, wherein the functional portion comprisesthe amino acid sequences of SEQ ID NOs: 13-18.
 8. The isolated orpurified polypeptide of claim 7, wherein the portion comprises the aminoacid sequences of SEQ ID NOs: 19 and
 20. 9. The isolated or purifiedpolypeptide of claim 8, comprising: a) SEQ ID NO: 23 and 24; or b) SEQID NO: 25 and
 26. 10. An isolated or purified protein comprising a firstpolypeptide chain comprising the amino acid sequences of SEQ ID NOs:13-15 and SEQ ID NO: 21 and a second polypeptide chain comprising theamino acid sequences of SEQ ID NOs: 16-18 and SEQ ID NO: 22, wherein theprotein is a human/mouse chimeric protein.
 11. The isolated or purifiedprotein of claim 10, wherein the first polypeptide chain comprises SEQID NO: 19 and the second polypeptide chain SEQ ID NO:
 20. 12. Theisolated or purified protein of claim 11, wherein the first polypeptidechain comprises SEQ ID NO: 25 and the second polypeptide chain comprisesSEQ ID NO:
 26. 13. The isolated or purified protein of claim 10, whereinthe protein is a fusion protein.
 14. The isolated or purified protein ofclaim 10, wherein the protein is a recombinant antibody.
 15. Acomposition comprising the TCR of claim 1 and a pharmaceuticallyacceptable carrier.
 16. A composition comprising the TCR of claim 5 anda pharmaceutically acceptable carrier.
 17. A composition comprising theTCR of claim 6 and a pharmaceutically acceptable carrier.
 18. Acomposition comprising the polypeptide of claim 7 and a pharmaceuticallyacceptable carrier.
 19. A composition comprising the polypeptide ofclaim 8 and a pharmaceutically acceptable carrier.
 20. A compositioncomprising the polypeptide of claim 9 and a pharmaceutically acceptablecarrier.
 21. A composition comprising the protein of claim 10 and apharmaceutically acceptable carrier.
 22. A composition comprising theprotein of claim 11 and a pharmaceutically acceptable carrier.
 23. Acomposition comprising the protein of claim 12 and a pharmaceuticallyacceptable carrier.